Gu X H, Casley D, Nayler W
Department of Medicine, University of Melbourne, Austin Hospital, Heidelberg, Victoria, Australia.
Eur J Pharmacol. 1989 Aug 22;167(2):281-90. doi: 10.1016/0014-2999(89)90589-x.
125I-labelled porcine endothelin (125I-endothelin) was used to identify specific high affinity endothelin binding sites in rat cardiac membrane fragments. Binding was to a single population of sites, with a KD of 0.20 +/- 0.03 nM and a Bmax of 93.5 +/- 6.4 fmol/mg protein at 37 degrees C. Reducing the temperature to 25 degrees C increased (P less than 0.02) the KD without changing Bmax. 125I-Endothelin binding was Ca2+ independent. Specific binding was saturable and displaceable by cold endothelin and sarafotoxin S6b, but not by (-)Bay K8644, nicardipine, (-)D888, (+)cis-diltiazem, prenylamine, lidoflazine, flunarizine, nor by 10(-10)-10(-4) M CoCl2, nor 10(-10)-10(-4) M NiCl2. omega-Conotoxin, prazosin, isoprenaline, angiotensin II and its inhibitor, vasopressin and its inhibitor, glyceryl trinitrate, amiloride, ergometrine and FII stonefish toxin also failed to displace bound 125I-endothelin. 10(-4)-10(-2) M CaCl2, 10(-4)-10(-2) M MgCl2, 3 X 10(-6)-10(-3) M MnCl2, 10(-5)-3 X 10(-4) M NiCl2, and 3 X 10(-5)-3 X 10(-4) M CoCl2 stimulated the binding. Incubation at 100 degrees C for 10 min destroyed specific binding.
用¹²⁵I标记的猪内皮素（¹²⁵I - 内皮素）来鉴定大鼠心肌膜片段中的特异性高亲和力内皮素结合位点。结合作用于单一的位点群体，在37℃时，解离常数（KD）为0.20±0.03 nM，最大结合容量（Bmax）为93.5±6.4 fmol/mg蛋白质。将温度降至25℃会使KD增加（P＜0.02），而Bmax不变。¹²⁵I - 内皮素的结合不依赖于Ca²⁺。特异性结合是可饱和的，并且可被冷内皮素和芋螺毒素S6b取代，但不能被（ - ）Bay K8644、尼卡地平、（ - ）D888、（ + ）顺式地尔硫䓬、普尼拉明、利多氟嗪、氟桂利嗪取代，也不能被10⁻¹⁰ - 10⁻⁴ M CoCl₂或10⁻¹⁰ - 10⁻⁴ M NiCl₂取代。ω - 芋螺毒素、哌唑嗪、异丙肾上腺素、血管紧张素II及其抑制剂、血管加压素及其抑制剂、硝酸甘油、阿米洛利、麦角新碱和石鱼毒素FII也不能取代结合的¹²⁵I - 内皮素。10⁻⁴ - 10⁻² M CaCl₂、10⁻⁴ - 10⁻² M MgCl₂、3×10⁻⁶ - 10⁻³ M MnCl₂、10⁻⁵ - 3×10⁻⁴ M NiCl₂和3×10⁻⁵ - 3×10⁻⁴ M CoCl₂能刺激结合。在100℃孵育10分钟会破坏特异性结合。
